We will apply a new class of biomarker harvesting nanocage technology to create a highly sensitive, multiplex, urine and blood test that uses mass spectrometry (MS) to attain absolute specificity for Babesia species causing human babesiosis and other tick borne pathogens. Babesiosis, a malaria like illness caused by intraerythrocytic protozoan species of the genus Babesia, is a rising medical problem in the United States, and still poses unsolved diagnostic issues. In the most severe cases, Babesia infection is associated with thrombocytopenia, unstable blood pressure, acute respiratory distress, disseminated intravascular coagulation, renal and liver failure, changes in brain functions, and death. Microscopy diagnosis is accurate only in acute, symptomatic patients, and requires manual, non-automated review of multiple blood smears. An accurate, faster and more economical direct antigen test is urgently needed. Since the course of Babesiosis is elevated in severity in the presence of concurrent tick borne infections, a test that, in a single sample, can detect coinfections with great sensitivity and specificity would be of great value for patient management. A sensitive and specific protein based direct test for screening US blood supplies is lacking to guide rejection of donors only in presence of active disease, in contrast with currently used indirect serological methods. As shown by our supporting data, we achieved the high levels of analytical sensitivity necessary to detect hundreds of babesial proteins in lysed red blood cells, plasma and urine of Babesia microti infected Golden hamsters and infected patients. This has been impossible in the past because of the extreme low abundance of babesial markers in the biomolecule matrix of blood and urine. Our nanocage nanoparticles can capture, concentrate, and preserve, biomolecules with high affinity, increasing the effective sensitivity greater than 100 fold, reduce the background, achieving a yield close to 100 percent. We propose to detect and measure specific Babesia protein antigens in the blood and urine of infected Golden hamsters and humans at a sensitivity of 5 picograms per mL. Unique chemical baits, immobilized in the Nanotraps are modified organic dyes that bind proteins, nucleic acids, and glycans, with extremely high affinity (KD <= 10-13 M) and a very low off-rate. We will uniquely identify markers shed by active pathogens belonging to the following families: Borreliaceae (e.g. Borrelia burgdorferi sl, Borrelia hermsii), Bartonella henselae, Rickettsiales, Ehrlichiaceae, Mycoplasma, Anaplasma phagocytophilum, Francisella tularensis, Powassan virus, Tick borne encephalitis virus, Colorado tick fever virus using a novel bioinformatics pipeline. Successful clinical validation of a novel direct urine and blood test, as proposed here, has broad implications for babesiosis and tick borne disease screening, transmission control, and treatment management. If successful, this study will provide scientific evidence to support the use of tandem MS for bacteria speciation in host samples without prior culture and validate a panel of Babesia and other pathogen antigens that can be used in the future as epitopes to develop more specific and sensitive diagnostic immunoassays.